Blood phosphorylated (p)-tau 181 and p-tau 217 have been proposed as accurate biomarkers of Alzheimer’s disease (AD) pathology. However, blood p-tau 181 is also elevated in amyotrophic lateral sclerosis (ALS) without a clearly identified source. We measured serum p-tau 181 and p-tau 217 in a multicentre cohort of ALS (n = 152), AD (n = 111) cases and disease controls (n = 99) recruited from four different centres. Further, we investigated the existence of both p-tau species using immunohistochemistry (IHC) and mass spectrometry (MS) in muscle biopsies of ALS cases (IHC: n = 13, MS: n = 5) and disease controls (IHC: n = 14, MS: n = 5) from one cohort. Serum p-tau 181 and p-tau 217 were higher in AD and ALS patients compared to disease controls. IHC and MS analyses revealed the presence of p-tau 181 and 217 in muscle biopsies from both ALS cases and disease controls, with ALS samples showing increased p-tau reactivity in atrophic muscle fibres. Blood p-tau species could potentially be used to diagnose both ALS and AD.

The application of quantitative proteomics provides a new and promising tool for standardized toxicological research. However, choosing a suitable quantitative method still puzzles many researchers because the optimal method needs to be determined. In this study, we investigated the advantages and limitations of two of the most commonly used global quantitative proteomics methods, namely label-free quantitation (LFQ) and tandem mass tags (TMT). As a case study, we exposed hepatocytes (HepG2) to the environmental contaminant benzo[a]pyrene (BaP) using a concentration of 2 µM. Our results revealed that both methods yield a similar proteome coverage, in which for LFQ a wider range of fold changes was observed but with less significant p-values compared to TMT. We detected 37 and 47 significantly enriched pathways by LFQ and TMT, respectively, with 17 overlapping pathways. To define the minimally required effort in proteomics as a benchmark, we artificially reduced the LFQ, and TMT data sets stepwise and compared the pathway enrichment. Thereby, we found that fewer proteins are necessary for detecting significant enrichment of pathways in TMT compared to LFQ, which might be explained by the higher reproducibility of the TMT data that was observed. In summary, we showed that the TMT approach is the preferable one when investigating toxicological questions because it offers a high reproducibility and sufficient proteome coverage in a comparably short time.

Sensing microbial tryptophan catabolites by the aryl hydrocarbon receptor (AhR) plays a pivotal role in host-microbiome homeostasis by modulating the host immune response. Nevertheless, the involved cellular processes triggered by the metabolites are mainly unknown. Here, we analyzed proteomic changes in macrophages after treatment with the tryptophan metabolites indole-3-acetic acid (I3AA) or indole-3-aldehyde (IAld), as well as the prototypic exogenous AhR-ligand benzo(a)pyrene (BaP) in the absence and presence of lipopolysaccharide (LPS) to identify affected cellular processes and pathways. The AhR-ligands regulated metabolic and immunologic processes in dependency of LPS co-stimulation. All investigated ligands time-dependently enhanced fatty acid β-oxidation. Differences due to the combination with LPS were observed for all three ligands. Additionally, oxidative phosphorylation was significantly increased by IAld and I3AA in a time and LPS-dependent manner. Immunoregulatory processes were affected in distinct ways. While BaP and I3AA up-regulated IL-8 signaling, IL-6 signaling was decreased by IAld. BaP decreased the inflammasome pathway. Thus, AhR-ligand-dependent regulations were identified, which may modulate the response of macrophages to bacterial infections, but also the commensal microbiota through changes in immune cell signaling and metabolic pathways that may also alter functionality. These findings highlight the relevance of AhR for maintaining microbial homeostasis and, consequently, host health.

Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.